PROJECT SUMMARY/ABSTRACT T lymphocytes comprise two major lineages, ?? and ??, which play critical, partially-distinct roles in host defense. ?? and ?? T cells arise from a common progenitor in the thymus; however, the molecular processes responsible for specification of these lineages during development in the thymus remain unclear and this represents a major gap in knowledge. We seek to address this knowledge gap. In doing so, we have provided compelling evidence that specification of the ?? and ?? T cell fates is controlled by differences in T cell receptor (TCR) signal strength. These signaling differences regulate fate by proportional induction of Id3, which causes graded repression of the function of E box DNA binding proteins (E proteins; E2A and HEB). To gain insight into how E protein binding to the genome is remodeled during fate specification, we (all Projects) employed a comprehensive, genome-wide approach, which revealed a number of important insights. First, the repression of E protein family members by strong TCR signals is selective, in that E2A binding to the genome is markedly repressed, while HEB binding is preserved (all Projects). Importantly, the sparing of HEB binding appears to be important, as HEB function is required for development of ?? T cells that produce IL-17 (Proj1/3/4). Second, E protein binding is closely associated with non-coding transcription, including long non-coding RNAs (lncRNA; all Projects). Indeed, we (Projects 1 and 4) demonstrated that one such lncRNA, ThymoD, plays an essential role in T lineage commitment. Finally, from among the numerous regulatory elements whose occupancy by E proteins is modulated during ?? lineage commitment, we have already identified two that play critical roles in controlling ?? development and function. Indeed, disruption of E protein binding to regulatory elements controlling the expression of either transcription factor Tcf7 or lncRNA Gm15417 enhances ?? T cell maturation and promotes adoption of the IL-17 producing effector fate. Consequently, in the current proposal, we integrate all of these insights to elucidate the mechanistic basis by which changes in E protein binding to these elements controls development of IL-17 producing ?? T cells. Insight into the factors controlling development of IL-17 producing ?? T cells is critical, as these cells have been implicated in numerous immune-mediated pathologies and cancer progression. To execute this research plan, we are entirely dependent upon the expertise of the Project Leaders in the program, as well as Genomics Core B. This effort promises to provide great insight into the role of E proteins in controlling both ?? development and function, which is of critical importance to human health and disease.